Saswati N. Chand

HuR is a post-transcriptional regulator of core metabolic enzymes in pancreatic cancer

Cancer cell metabolism differs from normal cells, yet the regulatory mechanisms responsible for these differences are incompletely understood, particularly in response to acute changes in the tumor microenvironment. HuR, an RNA-binding protein, acts under acute stress to regulate core signaling pathways in cancer through post-transcriptional regulation of mRNA targets. We demonstrate that HuR regulates the metabolic phenotype in pancreatic cancer cells and is critical for survival under acute glucose deprivation. Using three pancreatic cancer cell line models, HuR-proficient cells demonstrated superior survival under glucose deprivation when compared with isogenic cells with siRNA-silencing of HuR expression (HuR-deficient cells). We found that HuR-proficient cells utilized less glucose, but produced greater lactate, as compared with HuR-deficient cells. Acute glucose deprivation was found to act as a potent stimulus for HuR translocation from the nucleus to the cytoplasm, where HuR stabilizes its mRNA targets. We performed a gene expression array on ribonucleoprotein-immunoprecipitated mRNAs bound to HuR and identified 11 novel HuR target transcripts that encode enzymes central to glucose metabolism. Three (GPI, PRPS2 and IDH1) were selected for validation studies, and confirmed as bona fide HuR targets. These findings establish HuR as a critical regulator of pancreatic cancer cell metabolism and survival under acute glucose deprivation. Further explorations into HuR’s role in cancer cell metabolism should uncover novel therapeutic targets that are critical for cancer cell survival in a metabolically compromised tumor microenvironment.

HuR Contributes to TRAIL Resistance by Restricting Death Receptor 4 Expression in Pancreatic Cancer Cells

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal cancers, in part, due to resistance to both conventional and targeted therapeutics. TRAIL directly induces apoptosis through engagement of cell surface Death Receptors (DR4 and DR5), and has been explored as a molecular target for cancer treatment. Clinical trials with recombinant TRAIL and DR-targeting agents, however, have failed to show overall positive outcomes. Herein, we identify a novel TRAIL resistance mechanism governed by Hu antigen R (HuR, ELAV1), a stress-response protein abundant and functional in PDA cells. Exogenous HuR overexpression in TRAIL-sensitive PDA cell lines increases TRAIL resistance whereas silencing HuR in TRAIL-resistant PDA cells, by siRNA oligo-transfection, decreases TRAIL resistance. PDA cell exposure to soluble TRAIL induces HuR translocation from the nucleus to the cytoplasm. Furthermore, it is demonstrated that HuR interacts with the 3′-untranslated region (UTR) of DR4 mRNA. Pre-treatment of PDA cells with MS-444 (Novartis), an established small molecule inhibitor of HuR, substantially increased DR4 and DR5 cell surface levels and enhanced TRAIL sensitivity, further validating HuRs role in affecting TRAIL apoptotic resistance. NanoString analyses on the transcriptome of TRAIL-exposed PDA cells identified global HuR-mediated increases in antiapoptotic processes. Taken together, these data extend HuRs role as a key regulator of TRAIL-induced apoptosis. Implications: Discovery of an important new HuR-mediated TRAIL resistance mechanism suggests that tumor-targeted HuR inhibition increases sensitivity to TRAIL-based therapeutics and supports their re-evaluation as an effective treatment for PDA patients. Mol Cancer Res; 14(7); 599–611. ©2016 AACR.

WEE1 inhibition in pancreatic cancer cells is dependent on DNA repair status in a context dependent manner

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease, in part, because of the lack of effective targeted therapeutic options. MK-1775 (also known as AZD1775), a mitotic inhibitor, has been demonstrated to enhance the anti-tumor effects of DNA damaging agents such as gemcitabine. We evaluated the efficacy of MK-1775 alone or in combination with DNA damaging agents (MMC or oxaliplatin) in PDA cell lines that are either DNA repair proficient (DDR-P) or deficient (DDR-D). PDA cell lines PL11, Hs 766T and Capan-1 harboring naturally selected mutations in DNA repair genes FANCC, FANCG and BRCA2 respectively, were less sensitive to MK-1775 as compared to two out of four representative DDR-P (MIA PaCa2 and PANC-1) cell lines. Accordingly, DDR-P cells exhibit reduced sensitivity to MK-1775 upon siRNA silencing of DNA repair genes, BRCA2 or FANCD2, compared to control cells. Only DDR-P cells showed increased apoptosis as a result of early mitotic entry and catastrophe compared to DDR-D cells. Taken together with other recently published reports, our results add another level of evidence that the efficacy of WEE1 inhibition is influenced by the DNA repair status of a cell and may also be dependent on the tumor type and model evaluated.

Posttranscriptional Upregulation of IDH1 by HuR Establishes a Powerful Survival Phenotype in Pancreatic Cancer Cells

Cancer aggressiveness may result from the selective pressure of a harsh nutrient-deprived microenvironment. Here we illustrate how such conditions promote chemotherapy resistance in pancreatic ductal adenocarcinoma (PDAC). Glucose or glutamine withdrawal resulted in a 5- to 10-fold protective effect with chemotherapy treatment. PDAC xenografts were less sensitive to gemcitabine in hypoglycemic mice compared with hyperglycemic mice. Consistent with this observation, patients receiving adjuvant gemcitabine (n = 107) with elevated serum glucose levels (HgbA1C > 6.5%) exhibited improved survival. We identified enhanced antioxidant defense as a driver of chemoresistance in this setting. ROS levels were doubled in vitro by either nutrient withdrawal or gemcitabine treatment, but depriving PDAC cells of nutrients before gemcitabine treatment attenuated this effect. Mechanistic investigations based on RNAi or CRISPR approaches implicated the RNA binding protein HuR in preserving survival under nutrient withdrawal, with or without gemcitabine. Notably, RNA deep sequencing and functional analyses in HuR-deficient PDAC cell lines identified isocitrate dehydrogenase 1 (IDH1) as the sole antioxidant enzyme under HuR regulation. HuR-deficient PDAC cells lacked the ability to engraft successfully in immunocompromised mice, but IDH1 overexpression in these cells was sufficient to fully restore chemoresistance under low nutrient conditions. Overall, our findings highlight the HuR-IDH1 regulatory axis as a critical, actionable therapeutic target in pancreatic cancer. Cancer Res; 77(16); 4460-71. ©2017 AACR.

CRISPR Knockout of the HuR Gene Causes a Xenograft Lethal Phenotype

Pancreatic ductal adenocarcinoma (PDA) is the third leading cause of cancer-related deaths in the United States, whereas colorectal cancer is the third most common cancer. The RNA-binding protein HuR (ELAVL1) supports a pro-oncogenic network in gastrointestinal (GI) cancer cells through enhanced HuR expression. Using a publically available database, HuR expression levels were determined to be increased in primary PDA and colorectal cancer tumor cohorts as compared with normal pancreas and colon tissues, respectively. CRISPR/Cas9 technology was successfully used to delete the HuR gene in both PDA (MIA PaCa-2 and Hs 766T) and colorectal cancer (HCT116) cell lines. HuR deficiency has a mild phenotype, in vitro, as HuR-deficient MIA PaCa-2 (MIA.HuR-KO(−/−)) cells had increased apoptosis when compared with isogenic wild-type (MIA.HuR-WT(+/+)) cells. Using this isogenic system, mRNAs were identified that specifically bound to HuR and were required for transforming a two-dimensional culture into three dimensional (i.e., organoids). Importantly, HuR-deficient MIA PaCa-2 and Hs 766T cells were unable to engraft tumors in vivo compared with control HuR-proficient cells, demonstrating a unique xenograft lethal phenotype. Although not as a dramatic phenotype, CRISPR knockout HuR HCT116 colon cancer cells (HCT.HuR-KO(−/−)) showed significantly reduced in vivo tumor growth compared with controls (HCT.HuR-WT(+/+)). Finally, HuR deletion affects KRAS activity and controls a subset of pro-oncogenic genes. Implications: The work reported here supports the notion that targeting HuR is a promising therapeutic strategy to treat GI malignancies. Mol Cancer Res; 15(6); 696–707. ©2017 AACR.

Studying RNA-Binding Protein Interactions with Target mRNAs in Eukaryotic Cells: Native Ribonucleoprotein Immunoprecipitation (RIP) Assays

Post-transcriptional regulation of mRNA can potently dictate protein expression patterns in eukaryotic cells. This mode of regulation occurs through cis-acting regulatory regions in the mRNA transcript that mediate direct interactions with trans-acting RNA-binding proteins (RBPs). This mRNA/protein interaction can be studied in numerous ways that range from in vitro to in vivo through messenger ribonucleoprotein immunoprecipitation (mRNP-IP or RIP) assays. This modified immunoprecipitation approach is an important and sensitive method to determine the regulation of gene expression by specific RBPs under different cellular stressors.

RNA binding protein HuR regulates extracellular matrix gene expression and pH homeostasis independent of controlling HIF-1α signaling in nucleus pulposus cells

Nucleus pulposus (NP) cells reside in the hypoxic niche of the intervertebral disc. Studies have demonstrated that RNA-binding protein HuR modulates hypoxic signaling in several cancers, however, its function in the disc is unknown. HuR did not show cytoplasmic translocation in hypoxia and its silencing did not alter levels of Hif-1α or HIF-targets in NP cells. RNA-Sequencing data revealed that important extracellular matrix-related genes including several collagens, MMPs, aggrecan, Tgf-β3 and Sdc4 were regulated by HuR. Further analysis of HuR-silenced NP cells confirmed that HuR maintained expression of these matrix genes. We confirmed decreased levels of secreted collagen I and Sdc4 and increased pro-MMP13 in HuR-knockdown cells. In addition, messenger ribonucleoprotein immunoprecipitation demonstrated HuR binding to Tgf-β3 and Sdc4 mRNAs. Interestingly, while HuR bound to Hif-1α and Vegf mRNAs, it was clear that compensatory mechanisms sustained their expression when HuR was silenced. Noteworthy, despite the presence of multiple HuR-binding sites and reported interaction in other cell types, HuR showed no binding to Pgk1, Eno1, Pdk1 and Pfkfb3 in NP cells. Metabolic studies showed a significant decrease in the extracellular acidification rate (ECAR) and mitochondrial oxygen consumption rate (OCR) and acidic pH in HuR-silenced NP cells, without appreciable change in total OCR. These changes were likely due to decreased Ca12 expression in HuR silenced cells. Taken together, our study demonstrates for the first time that HuR regulates extracellular matrix (ECM) and pH homeostasis of NP cells and has important implications in the maintenance of intervertebral disc health.

Abstract 2854: CRISPR knockout of HuR in pancreatic cancer cells causes a xenograft lethal phenotype

Pancreatic ductal adenocarcinoma (PDA) is the most prevalent type of pancreatic cancer and will soon become the second leading cause of cancer related deaths in the U.S. Studies show that the nuclear localized mRNA-binding protein HuR (ELAVL1) is activated in PDA cells, with cytoplasmic translocation associated with increased tumor size and poor prognosis. Previous in vitro and in vivo studies have established HuR9s role as a PDA cell survival mechanism. Thus, we explored the phenotypic effect of completely eliminating HuR expression from PDA cells through the use of clustered, regularly interspaced, short palindromic repeat (CRISPR)/Cas9 technology to target and disrupt the HuR genomic sequence. Since INDELs are induced randomly, we designed 3 gRNAs to target HuR at different loci. Gene disruption was determined via sequencing and validated through protein and mRNA expression, where homozygous knockouts (HuR −/− ) had undetectable HuR expression as compared to wild-type (HuR +/+ ), heterozygotes (HuR +/− ), and CRISPR/Cas9 negative control. Sanger sequencing confirmed homozygous knockouts with a frame shift mutation on both alleles. When HuR knockout cells were exposed to chemotherapeutic stress including mitomycin C, oxaliplatin, and gemcitabine, no HuR expression (nuclear or cytoplasmic) was detected via immunofluorescence. Phenotypically, HuR −/− cells resulted in increased apoptosis and necrosis as measured via trypan blue assay, and accordingly, had increased caspase 3 activity, a marker of a cell death. HuR −/− cells, when treated with mitomycin C, oxaliplatin, gemcitabine, and glucose deprivation exhibited decreased long and short-term cell survival as compared to control cells. HuR −/− cells, pulse-labeled with bromodeoxyurdine (BrdU), had a higher proportion of cells in S phase and fewer cells in G2/M phase. HuR deletion enhanced premature mitotic entry thereby preventing efficient repair of DNA damage, leading to cell death. Importantly, CRISPR knockout of HuR showed marked impairment in tumor growth in mouse xenografts. The differences in median tumor volume with HuR −/- xenografts was significant as compared to xenografts in mice with HuR (+/+) cells (0.0 mm 3 vs 378.0 mm 3 , P Citation Format: Edwin Cheung, Shruti Lal, Mahsa Zarei, Nicole C. Mambelli-Lisboa, Saswati Chand, Carmella Romeo, Kevin O’Hayer, Eric Londin, Joseph A. Cozzitorto, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. CRISPR knockout of HuR in pancreatic cancer cells causes a xenograft lethal phenotype. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2854.

Abstract 5476: A novel PARP inhibitor resistance mechanism mediated by the RNA-binding protein HuR

Introduction: Pancreatic ductal adenocarcinoma (PDA) is the 4th leading cause of cancer-related deaths in the United States, and the 3rd most common cancer associated with BRCA mutations. Frontline therapies have significant toxicities and only minimally extend overall survival, highlighting the need to optimize targeted therapies. Poly-ADP ribose polymerase (PARP) inhibitors (PARPi), a ‘poster child’ for personalized medicine, depend on the concept of synthetic lethality where the combined perturbation of DNA repair genes, via genetic mutations within the tumor cells, and pharmacological PARP inhibition effectively targets BRCA-deficient tumors. Although PARPi have delivered promising preclinical and clinical results, initially- responsive patients ultimately develop resistance. A unique mechanism elucidated by our lab demonstrates that the mRNA-binding protein HuR mediates resistance to DNA damaging agents through post-transcriptional regulation of select mRNA cargo. Predominantly expressed in the nucleus, HuR translocates to the cytoplasm upon tumor-associated stress. Cytoplasmic HuR binds and stabilizes unique pro-survival transcripts, resulting in resistance to a harsh tumor microenvironment. Here, we sought to evaluate the role of HuR in regulating PARPi response. Methods and Results: Through immunofluorescence and western blot of fractionated lysates, we demonstrate that the PARP inhibitors Veliparib, Olaparib, and Rucaparib induced cytoplasmic HuR localization. Conversely, pre-treatment with MS-444 (Novartis), an established small molecule inhibitor of HuR, abrogated its nuclear export induced by PARPi treatment. Consistent with these findings, the growth-inhibitory effects of PARPi treatment were significantly potentiated upon HuR silencing whereas ectopic HuR overexpression promoted resistance, as observed in short term cell survival and long-term anchorage-independent growth assays. Additionally, silencing of HuR enhanced PARPi-induced cytotoxicity, assessed by increased accumulation of DNA damage (γH2Ax) foci and Poly ADP-ribose (PAR) polymers. Ribonucleotide protein immunoprecipitation (RNP-IP) assays demonstrated that HuR binds and upregulates Poly-ADP Ribose Glycohydrolase (PARG) mRNA, the major enzyme responsible for catabolism of PAR. Taken together, when PDA cells are exposed to PARPi, HuR mediates upregulation of PARG, thereby decreasing PARylation and facilitating DNA repair. Conversely, HuR inhibition results in detrimental accumulation of PAR and enhanced DNA damage, which ultimately leads to increased PARPi-conferred cytotoxicity. Discussion: These results demonstrate that HuR imposes a significant barrier to PARPi therapy by orchestrating a strong chemoresistance mechanism. Thus, we provide evidence that HuR (and/or its target) inhibition via an HuR inhibitor (MS-444) can optimize PARPi-based therapies for better patient outcomes. Citation Format: Saswati N. Chand, Akshay R. Kamath, Nicole Meisner-Kober, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. A novel PARP inhibitor resistance mechanism mediated by the RNA-binding protein HuR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5476. doi:10.1158/1538-7445.AM2015-5476

Abstract 1656: MK-1775 (WEE1 inhibition) lacks efficacy against DNA repair deficient pancreatic cancer cells

Introduction: In this study, we explored the possibility of developing a personalized approach of using MK-1775, a potent WEE1 inhibitor, as mono- or combination therapy to treat pancreatic ductal adenocarcinoma (PDA) cells with DNA repair deficiency. Experimental Methods: We treated PDA cells harboring diverse genetic backgrounds with IC50 doses of clinically-relevant DNA damaging agents: mitomycin C (MMC) and the WEE1 inhibitor MK-1775. Drug sensitivity assays were performed in isogenic PDA cells that are either proficient or deficient in Fanconi Anemia (FA)/BRCA2 pathway. In addition, proficient cell lines were treated with siRNA oligos targeted against FANCD2 and BRCA2 genes. Mechanistic studies such as Annexin V staining were performed to measure apoptotic cells upon MMC and MK-1775 treatments. The degree of DNA damage was measured by immunofluorescence (IF) assay using γH2AX antibody. Mitotic entry was analyzed by both IF and flow cytometry analyses using pH3 antibody. Results: Drug sensitivity assays demonstrated that FA/BRCA2-pathway proficient PDA cell lines (MiaPaCa2 and Panc1) are sensitive to the cytotoxic effect of MK-1775 compared to DNA repair-deficient cell lines (Capan1:BRCA2 deficient and Hs766T:FANCG deficient) which showed acute resistance to MK-1775. Immunoblotting showed that MK-1775 efficiently reduces the expression of WEE1 and accordingly phosphorylation of CDK1 in all cell lines. Annexin V staining showed a higher percentage of cell death in the FA/BRCA2-pathway proficient cell lines compared to deficient cell lines when exposed to MK-1775. Furthermore, IF experiments demonstrated that MMC treatment induces γH2AX foci in all cell lines, however, the FA/BRCA2 proficient cell lines showed a higher degree of nuclear abnormality and multi-nucleation after MK-1775 treatment compared to deficient cell lines. In addition, the FA/BRCA2 proficient cell lines showed significantly higher phospho histone 3 (pH3) staining, a marker of mitotic cells. FACS analyses validated that proficient cell lines showed a higher percentage of cells in the mitotic phase when exposed to MK-1775. In addition, we evaluated the cytotoxic effect of MK-1775 in combination with MMC and showed that FA/BRCA2 proficient cells are more sensitive to dual treatment than deficient cells. In addition, immnobloting detected cleaved caspase 3, a marker of apoptosis, after MK-1775 treatment alone or in combination with MMC, demonstrating cells undergoing mitotic catastrophe. We validated the results in the FA/BRCA2-pathway proficient cell lines pre-treated with siFANCD2 or siBRCA2 oligos as compared to the proficient control lines. Conclusions: These results support a paradigm in which identified high risk FA/BRCA2-mutated patients would not benefit from WEE1 inhibitor monotherapy; and thus, would most likely respond better to conventional DNA damaging agent-based therapies (e.g., oxaliplatin or MMC). Citation Format: Shruti Lal, Saswati N. Chand, Emanuela Dylgjeri, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. MK-1775 (WEE1 inhibition) lacks efficacy against DNA repair deficient pancreatic cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1656. doi:10.1158/1538-7445.AM2015-1656